Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequencing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a periods of three times the SRT. One set was operated at an SRT of 2 days and another set was operated at an SRT of 8 days. Samples for T-RFLP analysis were collected from the two sets of replicate reactors. HhaI, MspI, and RsaI T-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis with Ward's method using Jaccard distance and Hellinger distance showed that the bacterial community structure in both sets of reactors from both experimental runs was dynamic and that replicate reactors were clustered together and evolved similarly from startup. Richness (S), evenness (E), the Shannon-Weaver index (H), and the reciprocal of Simpson's index (1/D) were calculated, and the values were compared between the two sets of reactors. Evenness values were higher for reactors operated at an SRT of 2 days. Statistically significant differences in diversity (H and D) between the two sets of reactors were tested using a randomization procedure, and the results showed that reactors from both experimental runs that were operated at an SRT of 2 days had higher diversity (H and D) at the 5% level. T-RFLP analysis with diversity indices proved to be a powerful tool to analyze changes in the bacterial community diversity in response to changes in the operational parameters of activated-sludge systems.

[1]  H. Odegaard,et al.  Coarse media filtration for enhanced primary treatment of municipal wastewater. , 2002, Water science and technology : a journal of the International Association on Water Pollution Research.

[2]  J. H. Ward.  Hierarchical Grouping to Optimize an Objective Function , 1963 .

[3]  Lawrence O. Ticknor,et al.  Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities , 2001, Applied and Environmental Microbiology.

[4]  Daniel B. Oerther,et al.  Bacterial Competition in Activated Sludge: Theoretical Analysis of Varying Solids Retention Times on Diversity , 2004, Microbial Ecology.

[5]  M. Schutter,et al.  Use of Length Heterogeneity PCR and Fatty Acid Methyl Ester Profiles To Characterize Microbial Communities in Soil , 2000, Applied and Environmental Microbiology.

[6]  안난희,et al.  Terminal Restriction Fragment Length Polymorphism ( T-RFLP ) 분석을 이용한 다양한 활성 슬러지의 세균 군집비교 , 2002 .

[7]  A. Hiraishi,et al.  Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge. , 2000, Journal of bioscience and bioengineering.

[8]  F. W. Gilcreas,et al.  Standard methods for the examination of water and waste water. , 1966, American journal of public health and the nation's health.

[9]  W. Sloan,et al.  Prokaryotic diversity and its limits: microbial community structure in nature and implications for microbial ecology. , 2004, Current opinion in microbiology.

[10]  Jason D. Hoeksema,et al.  Linking biodiversity to ecosystem function: implications for conservation ecology , 2000, Oecologia.

[11]  C. Kuske,et al.  Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis , 2000, Applied and Environmental Microbiology.

[12]  J. Huisman,et al.  Biodiversity of plankton by species oscillations and chaos , 1999, Nature.

[13]  U. Göbel,et al.  Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. , 1997, FEMS microbiology reviews.

[14]  G. Muyzer,et al.  Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis , 1999, Applied and Environmental Microbiology.

[15]  L. Øvreås,et al.  Prokaryotic Diversity--Magnitude, Dynamics, and Controlling Factors , 2002, Science.

[16]  A. E. Greenberg,et al.  Standard methods for the examination of water and wastewater : supplement to the sixteenth edition , 1988 .

[17]  D. Graham,et al.  Theoretical ecology for engineering biology. , 2003, Environmental science & technology.

[18]  T. Curtis,et al.  The comparison of the diversity of activated sludge plants , 1998 .

[19]  Franz J. Weissing,et al.  Oscillations and chaos generated by competition for interactively essential resources , 2002, Ecological Research.

[20]  D. Tilman.  THE ECOLOGICAL CONSEQUENCES OF CHANGES IN BIODIVERSITY: A SEARCH FOR GENERAL PRINCIPLES101 , 1999 .

[21]  W. Verstraete,et al.  Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. , 2002, FEMS microbiology ecology.

[22]  Franz J. Weissing,et al.  BIOLOGICAL CONDITIONS FOR OSCILLATIONS AND CHAOS GENERATED BY MULTISPECIES COMPETITION , 2001 .

[23]  G. Muyzer,et al.  Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology , 2004, Antonie van Leeuwenhoek.

[24]  K. Timmis,et al.  An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. , 2000, Environmental microbiology.

[25]  David M. Stamper,et al.  Bacterial Population Changes in a Membrane Bioreactor for Graywater Treatment Monitored by Denaturing Gradient Gel Electrophoretic Analysis of 16S rRNA Gene Fragments , 2003, Applied and Environmental Microbiology.

[26]  P. de Vos,et al.  Bioaugmentation of Activated Sludge by an Indigenous 3-Chloroaniline-Degrading Comamonas testosteroni Strain, I2gfp , 2000, Applied and Environmental Microbiology.

[27]  C. Morris,et al.  Microbial Biodiversity: Approaches to Experimental Design and Hypothesis Testing in Primary Scientific Literature from 1975 to 1999 , 2002, Microbiology and Molecular Biology Reviews.

[28]  Jean-Jacques Godon,et al.  Microbial 16S rDNA diversity in an anaerobic digester , 1997 .

[29]  E. Triplett,et al.  Automated Approach for Ribosomal Intergenic Spacer Analysis of Microbial Diversity and Its Application to Freshwater Bacterial Communities , 1999, Applied and Environmental Microbiology.

[30]  Y. Li,et al.  Species Diversity Improves the Efficiency of Mercury-Reducing Biofilms under Changing Environmental Conditions , 2002, Applied and Environmental Microbiology.

[31]  C. Grady,et al.  Microbial population dynamics in laboratory-scale activated sludge reactors. , 2002, Water science and technology : a journal of the International Association on Water Pollution Research.

[32]  C. Nakatsu,et al.  Stability of the bacterial communities supported by a seven-stage biological process treating pharmaceutical wastewater as revealed by PCR-DGGE. , 2002, Water research.

[33]  T Nishihara,et al.  Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals. , 2001, Ecotoxicology and environmental safety.

[34]  W. Bossert,et al.  The Measurement of Diversity , 2001 .

[35]  Hans H. Cheng,et al.  Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA , 1997, Applied and environmental microbiology.

[36]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[37]  C. Criddle,et al.  Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity. , 2003, BioTechniques.

[38]  Sang Joon Kim,et al.  A Mathematical Theory of Communication , 2006 .

[39]  C. E. SHANNON,et al.  A mathematical theory of communication , 1948, MOCO.

[40]  T. Marsh.  Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. , 1999, Current opinion in microbiology.

[41]  P. Hugenholtz,et al.  The use of 16S rDNA clone libraries to describe the microbial diversity of activated sludge communities , 1998 .

[42]  C. Kitts,et al.  Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. , 2001, Current issues in intestinal microbiology.

[43]  Andrew R. Solow,et al.  A simple test for change in community structure , 1993 .

[44]  K. Timmis,et al.  Thermal Gradient Gel Electrophoresis Analysis of Bioprotection from Pollutant Shocks in the Activated Sludge Microbial Community , 1999, Applied and Environmental Microbiology.

[45]  E. Smit,et al.  Detection of shifts in microbial community structure and diversity in soil caused by copper contamination using amplified ribosomal DNA restriction analysis , 1997 .

[46]  Shahid Naeem,et al.  Biodiversity enhances ecosystem reliability , 1997, Nature.